Identification of regions within the four small subunits of human replication factor C required for complex formation and DNA replication

Abstract

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are processivity factors for eukoryotic DNA polymerases δ and ε. RFC binds to a DNA primer end and loads PCNA onto DNA in an ATP-dependent reaction. The five RFC subunits p140, p40, p38, p37, and p36, all of which are required to form the active RFC complex, share regions of high homology including the defined RFC boxes II-VIII. RFC boxes III and V constitute a putative ATP binding site, whereas the function of the other conserved boxes is unknown. To study the individual subunits in the RFC complex and the role of the RFC boxes, deletion mutations were created in all subunits. Sequences close to the C terminus of each of the small subunits are required for formation of the five subunit complex. A N-terminal region of the small subunits, containing the RFC homology box II, plays a critical role in the function of these subunits, deletion of which reduces but does not abolish RFC activity in loading PCNA onto DNA and in supporting an RFC-dependent replication reaction. The N termini of p37 and p40, although highly homologous, are not interchangeable, suggesting unique functions for the individual subunits.